Optogenetic control of hypocretin neurons and stress The neural underpinnings of the response to acute stress involve activation of neurons producing corticotrophin releasing factor (CRF) and interactions between multiple homeostatic circuits. However, the details of these interactions are poorly understood. Hypocretin (Hcrt, also known as Orexin)-producing neurons in the lateral hypothalamus (LH) are important for maintaining arousal stability since loss of Hcrt function has been linked to narcolepsy in mouse, dog and human subjects. Hypocretin neurons are activated by acute stress, receive innervation from CRF terminals and are depolarized by CRF. Conversely, infusion of Hcrt-1 activates the hypothalamo-pituitary-adrenal (HPA) axis, and hypocretin receptor antagonists can block the release of ACTH induced by acute stress. Here we propose to use a newly developed optogenetic method to test whether the activity of hypocretin neurons is necessary and sufficient to activate the HPA axis. In the first aim, we will determine whether Hcrt neurons are necessary to activate the HPA axis by monitoring the acute stress response in Hcrt-deficient mice. In specific aim 2, we will determine whether the activity of Hcrt neurons is sufficient to induce a stress-like response by using an optogenetic approach. We will also test when is this activation required by using mice transduced with a lentivirus expressing a photoactivatable chloride channel in hypocretin cells. This technology will allow us to decipher the neural code of the hypocretin network that is associated with the stress response. In the third aim, we will test the functional connectivity of Hcrt. We will test whether the effects of photostimulation on the HPA axis are mediated directly by CRF signaling in the paraventricular hypothalamic nucleus, or whether the effect is indirect through other brain structures. The data collected in this revised proposal will enhance our understanding of the neural basis of the stress response with unprecedented temporal resolution and may lead to novel therapeutics for stress disorders and related diseases, as well as identify potential side effects for drugs that target the Hcrt system for other disorders. PUBLIC HEALTH RELEVANCE: The data collected in this research proposal will enhance our understanding of stress response and may lead to novel therapeutics for stress disorders and related diseases in the general public.